The activation of muscarinic acetylcholine receptors initiates a complex physiological response that involves a number of second messenger systems. We are studying muscarinic signal transduction in the murine neuroblastoma cell line N1E-115 and in CHO cells with the goal of understanding how the various second messenger signals are coordinated. Our experiments are designed to examine interactions between intermediates in the signal transduction process by manipulating the pathways at specific points by applying exogenous metabolites or pharmacologically altering the activity of key enzymes. We will be concentrating on the Ca signals due to influx release, the cGMP & AA signals, and changes in ionic currents. Our methods include video imaging of fluorescent indicators, voltage clamp, release of caged metabolites, microinjection, and permeabilization. Muscarinic agonists, and other ligands, initiate (Ca)i oscillations in N1E-115 cells. Experiments are proposed to investigate the mechanisms responsible for these temporally and spatially complicated Ca signals. A key experiment involves attempts to phase shift the oscillation with brief pulses of Ca, IP3, cGMP, and IP4 using voltage clamp, microinjection and photolysis of caged compounds. The goal is to identify rate limiting steps in the processes leading to oscillation. We found that Ca influx is required for stimulating cGMP production and that Ca release will not substitute for influx. Our hypothesis is that agonist elicits two Ca signals that are compartmentalized and have different effects on metabolism. Voltage clamp experiments will be done to study the modulation of Ca influx by agonist. N1E-115 cells express receptors for several transmitters and we found that the transmitters interact and produce a cross-facilitation effect that we call recruitment. We will study the mechanism of interaction between the messenger pathways activated by different transmitters. One hypothesis is that cGMP production is important for recruitment. We will also be studying signal transduction in CHO cell lines separately transfected with the m1, m2, m3, and m4 gene sequences. These lines provide powerful new preparations for studying the signaling pathways engaged by muscarinic agonists. The results of our studies should be generally applicable to the broad class of receptors whose effects are mediated through G proteins.